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For Discussion on Discussion Paper GMO 02/11
5 July 2011
Genetically Modified Organisms
(Control of Release) Ordinance Cap. 607
Expert Group
Risk Assessment and Disposal of
Live Recombinant Veterinary Vaccines
Purpose
This paper briefs members on the current status of live recombinant
veterinary vaccines and their possible adverse effect on the local biodiversity, and
invite members’ view on the recommendation for the disposal of the live recombinant
veterinary vaccines.
Background
2. Live recombinant veterinary vaccines are vaccines where a live
microorganism (bacteria or virus) has been modified to express its entire genome or a
portion of foreign RNA or DNA sequences or proteins and where the replicative
competent vector acts as a carrier and may itself act as a protective immunogen. The
vaccines are attenuated and genetically defined live vaccines, which have definite,
non-reverting mutations or deletions, for veterinary uses.
3. The attenuation was achieved through expressing the antigen-encoding
genes isolated from pathogenic strains in non-virulent strains. It can also be achieved
through deleting the disease-causing genes in the pathogenic strains, rendering them
non-virulent.
4. Live recombinant veterinary vaccines are GMOs subject to the regulation
under the Genetically Modified Organisms (Control of Release) Ordinance Cap.607
(the Ordinance). Currently, these vaccines have not been registered in Hong Kong as
pharmaceutical products under the Pharmacy and Poisons Ordinance Cap.138.
However, according to Section 36(1A) of the Pharmacy and Poisons Regulations Cap.
- 2 -
138A, such vaccines could be imported or administered without registration for the
purpose of treatment by a registered veterinary surgeon of a particular animal.
5. Vaccination with live microorganisms may lead to the shedding or
spreading of the administrated microorganisms into the environment. The shed
microorganisms may grow or reproduce and bring about adverse impact on the
environment. Therefore, administration or import with the purpose of administration
of live recombinant veterinary vaccines would in effect be considered as release of
GMOs into the environment which requires prior approval from the Director of
Agriculture, Fisheries and Conservation under the Ordinance.
Risk Assessment
6. In view of the rapid development in the production of live recombinant
veterinary vaccines and the potential application of such vaccines in Hong Kong, a
risk assessment was undertaken to assess the possible adverse biosafety effect of the
commercially available strains of live recombinant veterinary vaccines on the local
environment.
7. There are a number of potential adverse biosafety effects that could be
resulted from the administration of the live recombinant veterinary vaccines,
including establishment of an undesirable self-sustaining population, altered
pathogenicity or host range, horizontal gene transfer and recombination with other
virus/bacteria, reversion to virulence, possibility to spread to the environment and
effects on local host species.
8. Based on the risk assessment, it is concluded that the risk to the biological
diversity of the local environment posed by the live recombinant veterinary vaccines
is acceptable. The detailed risk assessment report is attached at Annex.
Disposal of Live Recombinant Veterinary Vaccines
9. Under the Ordinance, a person must not knowingly release any unapproved
GMO. Anyone who has control of a GMO knows that the GMO is unapproved and
has been released into the environment shall also inform the Director of the release.
Contravention of the above requirements may be liable to prosecution under the
Ordinance.
10. At present, live recombinant veterinary vaccines are not approved or
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exempted GMOs. Therefore, the above restrictions apply to these kinds of vaccines.
However, the risk assessment undertaken indicates that the risk posed by the vaccines
to the biological diversity of the local environment is acceptable. On the other hand,
it is necessary to cater for the need of application of veterinary vaccines in emergency
situations. It should be noted that the Pharmacy and Poisons Regulations (Cap.138A)
exempts the registration of veterinary vaccines to be used for the purpose of treatment
by a registered veterinary surgeon of a particular animal.
11. Section 46 of the Ordinance empowers the Secretary for the Environment to
exempt any GMO from the application of section 5 (Restrictions on release into
environment and maintenance of lives of GMOs), 7 (Restrictions on import of GMOs
intended for release into environment) or 23 (Restrictions on export of GMOs
intended for release into environment) if the Secretary is satisfied that the possible
adverse biosafety effect that may result from the exemption is acceptable or
manageable. The Ordinance also provides that an exemption may take effect
generally or for any purposes or by reference to any circumstances, and either
conditionally or unconditionally.
12. Considering the above, it is recommended to grant exemption to live
recombinant veterinary vaccines from the application of section 5 and 7 of the
Ordinance, provided that the live recombinant veterinary vaccines are registered with
the Pharmacy and Poisons Board, or imported/administrated for the purpose of
treatment by a registered veterinary surgeon of a particular animal.
Advice Sought
13. Members are invited to note the detail risk assessment report of live
recombinant veterinary vaccines at Annex and comment on the proposed exemption
of live recombinant veterinary vaccines under the Ordinance.
Agriculture, Fisheries and Conservation Department
June 2011
- 4 -
Annex
Risk Assessment Report
Live Recombinant Veterinary Vaccines
- 5 -
Purpose
Genetically modified or live recombinant veterinary vaccines are vaccines where a
live microorganism (bacteria or virus) has been modified to express entire genomes or
a portion of foreign RNA or DNA sequences or proteins and where the replicative
competent vector acts as a carrier and may itself act as a protective immunogen. The
vaccines are attenuated and genetically defined live vaccines, which have definite,
non-reverting mutations or deletions, for veterinary uses (1). In view of the rapid
development in the production of live recombinant veterinary vaccines and the
potential application of such vaccines in Hong Kong, a risk assessment is undertaken
to assess the possible adverse biosafety effect of the live recombinant veterinary
vaccines on the local environment.
This risk assessment report was prepared in accordance with Schedule 3 of the
Genetically Modified Organisms (Control of Release) Ordinance Cap.607 with
respect to the requirements on risk assessment on possible adverse biosafety effects of
GMOs on the local environment.
Identities of the GMOs
Based on the information from the Biosafety Clearing-House, there are 16 strains of
commercially available live recombinant veterinary vaccines. The target animals
and diseases of the vaccines are listed in Table 1 below:
Table 1: The commercially available live recombinant vaccines for veterinary uses
# Commercial Name Target
Animal Target Disease
1 Proteqflu / Proteqflu-TE Horse Equine Influenza / + Tetanus
2 Recombitek Equine Influenza Horse Equine Influenza
3 Recombitek Equine WNV Horse West Nile Virus
4 Recombitek Canine Distemper Dog Distemper
- 6 -
5 Purevax Recombinant
Leukaemia Vaccine
Cat Feline Leukaemia
6 Purevax Feline Rabies Cat Feline Rabies
7 Equilis StrepE Horse Streptococcus equi Infection (Strangles)
8 PreveNile Horse West Nile Virus
9 AviPro® Megan® Vac 1 Chicken Salmonella Infection
10 Poulvac ST Chicken Salmonella Infection
11 Vaxxitek HVT+IBD Chicken Infectious Bursal Disease
12 Innovax-ILT Chicken Marek's Disease
13 Innovax-ND-SB Chicken Marek's Disease, Newcastle Disease
14 Nobi-Porvac Aujeszky Pig Aujeszky Disease
15 Suvaxyn Aujeszky Pig Aujeszky Disease
16 Poulvac E. coli Chicken Pathogenic E. coli Infection
Besides, various similar strains of live recombinant veterinary vaccines have been
developed and approved for use in China.
Recipient Organisms
Canarypox Virus - (for Vaccines # 1 - 6)
Canarypox virus belongs to the avipoxvirus family. It is a large, enveloped, double
stranded DNA virus of which canary is the natural host (2). The ALVAC strain used in
the preparation of the vaccines listed above was developed from an infectious strain
attenuated by 200 serial passages on chick embryo fibroblasts and four successive
plaque purifications. Because it is non-replicative in mammals, and genetically and
physically stable, the ALVAC strain is considered as a ubiquitous vaccine vector with
high biosafety (3).
Streptococcus equi - (for Equilis StrepE)
It is the bacterial pathogen that causes the strangle disease in equine species (horses,
donkeys, mules and zebras). It normally does not impose any danger to humans or
other domestic species (4).
- 7 -
Human Yellow Fever (YF) Virus - (for PreveNile)
It is an arthropod borne virus of the Flaviviridae family and the disease it caused can
be fatal to human. Like other members of the Flaviviridae family, such as dengue and
Japanese encephalitis viruses, this virus is transmitted by competent mosquito vectors
(Aedes spp.) and is a risk in tropical parts of Africa and South America (5). The virus
strain 17D used as the parental organism is an attenuated vaccine strain that has been
widely used around the world, including Canada. This virus strain has no replication
activity in live mosquito cells (6).
Salmonella typhimurium - (for AviPro® Megan® Vac 1 and Poulvac ST)
It is a bacterial pathogen that can infect a variety of domestic animals including
chickens, horses, cattle, pigs, dogs and cats. It is also a leading cause of human
gastroenteritis. The parental strain χ3761 S. typhimurium UK-1 isolated from a
moribund horse in USA is virulent to young chicks and have an LD50 value of 3x103
colony forming units (CFU) for one-day-old chicks (7, 8).
Turkey Herpesvirus - (for Vaxxitek HVT+IBD, Innovax-ILT and Innovax-ND-SB)
Turkey Herpesvirus (HVT) (FC-126 strain) is an enveloped double-stranded DNA
virus that was originally isolated from domestic turkeys in the late1960s. It is a
non-pathogenic alphaherpesvirus and has been widely used as a vaccine against
Marek's Disease since the early 1970s, due to its antigenic relationship to Marek’s
Disease Virus (MDV) (9, 10).
Aujeszky’s Disease Virus - (for Nobi-Porvac Aujeszky and Suvaxyn Aujeszky)
Pseudorabies (Aujeszky's Disease) Virus is an alphaherpesvirus that naturally infects
pigs and causes the Aujeszky's Disease. The virus can also infect nearly all
domesticated (including cattle, sheep, goats, cats and dogs) and wild mammals.
However, it does not infect humans (11).
Escherichia coli - (for Poulvac E. coli)
Although non-pathogenic E. coli are normally found in the intestines of poultry,
certain strains of E. coli will generate extra-intestinal infections, or colibacillosis, in
- 8 -
chickens. Colibacillosis is frequently associated with poor animal husbandry, and is a
common secondary infection following bacterial or viral infection. The parental E.
coli strain was isolated from a clinical case of avian colibacillosis. This strain is
pathogenic to chickens, as it was shown to be invasive and capable of persisting for at
least five weeks in specific pathogen-free (SPF) chicks (48).
Donor Organisms
Equine Influenza Virus - (for Proteqflu/Proteqflu-TE and Recombitek Equine
Influenza)
The disease Equine Influenza caused by this virus is endemic to members of the
Equidae family (i.e. horses, donkeys, mules and zebras) (12).
West Nile Virus - (for Recombitek Equine WNV and PreveNile)
It can affect many different species of animals. Humans are also susceptible to this
virus. It is transmitted by bites of infected mosquitoes (13).
Canine Distemper Virus - (for Recombitek Canine Distemper)
It is a virus that infects dogs and other mammals, including ferrets and raccoons. But
humans are not affected by this virus (14).
Feline Leukaemia Virus - (for Purevax Recombinant Leukaemia Vaccine)
It is a retrovirus that prominently infects cats. It is a common cause of cancers and
various blood disorders in cats. There is no evidence showing that the virus can be
transmitted from cats to humans (15).
Feline Rabies Virus - (for Purevax Feline Rabies)
It is transmitted through the saliva of infected animals. Humans can be infected by
this virus (16).
Infectious Bursal Disease Virus - (for Vaxxitek HVT+IBD)
It principally affects chickens. But turkeys, ducks, and some other species of domestic
- 9 -
and wild birds can also be infected by this virus (17).
Laryngotracheitis Virus - (for Innovax-ILT)
Infectious Laryngotracheitis Virus causes respiratory diseases in chickens. It can also
infect turkeys, pheasants, peafowl and perhaps other avian species (18).
Newcastle Disease Virus - (for Innovax-ND-SB)
It causes a contagious and fatal disease that affects most species of birds (19).
Chicken Herpesvirus - (for Innovax-ND-SB)
Chicken Herpesvirus (Marek’s Disease Virus) is mainly found on chickens, but can
also affect pheasants, quails, game fowls and turkeys (20).
Vectors
ALVAC®
Vector for Vaccines # 1 - 6
It is derived from the non-disease causing Canarypox Virus and is considered to be
the ‘backbone’ of ALVAC®
vaccines. It is genetically stable and allows for the
insertion of large amounts of foreign DNA. It was demonstrated that the ALVAC®
vector is only capable of replication within certain avian species and not at all in
mammals. Studies showed that the abortive step to viral replication occurs early in the
life cycle of the virus with only early proteins being synthesized following infection
of a cell (21).
Vectors for PreveNile
The vaccine was constructed by a two-plasmid system. Plasmid YF5’3’IV WN prME
encodes the 5’ UTR of Yellow Fever capsid, West Nile Virus prM gene, 5’ end of West
Nile Virus E gene and the 3’ end of Yellow Fever NS5 and UTR. Plasmid YFM5.2
WN encodes the second half of West Nile Virus E gene and the non-structural genes
of Yellow Fever NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5. West Nile prME gene
fragments were amplified by RT-PCR and sub-cloned into the two-plasmid system by
overlap-extension PCR. Silent Eag I and Bsp EI sites were introduced for in vitro
- 10 -
ligation steps necessary to create a full-length cDNA before in vitro transcription.
Naked RNA initiates productive infection after transfection of a Vero cell line (22).
Vectors for AviPro® Megan® Vac 1
No vector was constructed specifically for this GMO. Bacteriophage P22HT int was
used for transduction with standard methods and media. A library of S. typhimurium
mutants was generated by random insertion of DNA transduced by the phage. Fusaric
acid selection was then used for the desired deletion mutations (7).
Vector for Equilis StrepE
The vaccine strain was constructed by the electroporation of gene knock-out
constructs and gene deletion (+1kb) constructs. Thus, the vaccine strain has no
vector-derived antibiotic resistance markers or other foreign DNA (23).
Vector for Vaxxitek HVT+IBD
The vaccine was generated by inserting an IBDV VP2 gene expression cassette,
which consists of a mammalian virus promoter/enhancer, DNA sequence encoding the
VP2 capsid protein of IBDV, and a mammalian virus polyadenylation signal, into the
HVT genome (10).
Vectors for Poulvac ST, Innovax-ILT & Innovax-ND-SB
Information is not available.
Vector for Poulvac E. coli
The mutant aroA gene was sub-cloned into a mobilized suicide vector (SacB,
pKNG101), which was transferred to the parental pathogenic E. coli strain via
conjugation (48, 49).
Insert and Modification
Proteqflu / Proteqflu-TE
The vaccines contain two vaccine strains vCP2242 and vCP1533. The
- 11 -
Haemagglutinin (HA) genes from Equine Influenza Virus A/equi-2/Ohio/03 (for
vCP2242) and Equine Influenza Virus A/equi-2/Newmarket/2/93 (for vCP1533) were
cloned into the ALVAC®
vector and subsequently introduced into ALVAC, the
non-disease causing strain of Canarypox Virus (24).
Haemagglutinin is a kind of antigenic glycoproteins found on the surface of influenza
virus. Glycoproteins are important integral membrane proteins that play an important
role in cell to cell interactions. Haemagglutinin is required by the virus to attach to
host cells. The host’s immune response is usually induced by such kinds of surface
antigens (25).
Recombitek Equine Influenza
The haemagglutinin coding sequence from Equine Influenza Virus (EIV), either strain
Kentucky/94 or Newmarket/2/93, was inserted into ALVAC (26).
Recombitek Equine WNV
Gene sequence from West Nile Virus was selected from those likely to stimulate
protective immunity in horses and cloned into the ALVAC® vector and subsequently
introduced into ALVAC (27).
Recombitek Canine Distemper
The haemagglutinin (HA) and fusion (F) protein genes from Canine Distemper Virus
(CDV), flanked by the Vaccinia Virus H6 promoter, are introduced into ALVAC (28).
Fusion protein is a kind of glycoproteins found in the envelope of the virus. It
mediates the fusion of the viral membrane with the host cell membrane (29).
Purevax Recombinant Leukaemia Vaccine
The env and gag genes and part of the pol gene obtained from the Glasgow strain of
Feline Leukaemia Virus, flanked by the Vaccinia Virus H6 promoter, are introduced
into ALVAC (30).
Env protein encoded by this gene is part of the viral envelope covering the viral core.
- 12 -
It is responsible for receptor binding and fusion with the host membrane. It can elicit
host’s antibody responses (29). The gene gag encodes for the structural protein which
forms the skeleton of the viral core (31). The pol gene encodes for the
RNA-dependent DNA polymerase. It is the enzyme produced by the RNA virus and is
responsible for converting the viral RNA genome into DNA, which can be integrated
into the host genome or used for protein production.
Vaccinia Virus H6 promoter is a DNA sequence essential for the transcription of the
recombinant protein. It is obtained from the double stranded DNA virus Vaccinia
Virus (30).
Purevax Feline Rabies
The rabies glycoprotein gene, flanked by the Vaccinia Virus H6 promoter, was
introduced into ALVAC (32).
The glycoprotein protein is an accessory protein that promotes cell fusion during
infection (33).
Equilis StrepE
The aroA gene was deleted from the disease-causing bacteria S. equi. The gene
encodes for the 5-enolpyruvylshikimate 3-phosphate synthase involved in the
biosynthesis of aromatic amino acids, which is required for the bacteria to grow (34).
PreveNile
The premembrane (prM) and envelope (Env) genes of the West Nile Virus were
introduced into a live attenuated Human Yellow Fever Virus (6).
The premembrane is important for the correct conformational folding of the envelope
(29).
AviPro® Megan® Vac 1
The cya and crp genes were deleted from the genome of χ3761 S. typhimurium UK-1
(7).
- 13 -
The cya gene encodes for adenylate cyclase and crp encodes for the cyclic adenosine
monophosphate (cAMP) receptor protein. Both are key enzymes involved in the
cAMP biosynthesis. cAMP is an important second messenger for intracellular signal
transduction and is essential for the proper cell functioning (16).
Poulvac ST
The aroA gene from S. typhimurium LT2 strain was mutated and transduced into a
wild type parental strain using bacteriophage (35).
Vaxxitek HVT+IBD
Turkey Herpesvirus (HVT) was modified by expressing the VP2 protein from
Infectious Bursal Disease Virus (IBDV) (10).
VP2 protein is the major core proteins of IBDV and is identified as the major antigen
to elicit hosts’ neutralizing antibodies (36).
Innovax-ILT
Genes (unknown) from Laryngotracheitis Virus were introduced into Turkey
Herpesvirus (37).
Innovax-ND-SB
An unknown gene from Newcastle Disease Virus and the SB-1 strain of Chicken
Herpesvirus serotype 2 were introduced into Turkey Herpesvirus (38)
Nobi-Porvac Aujeszky
The glycoprotein gI gene and thymidine kinase gene are deleted from Aujeszky's
Disease Virus (39).
Thymidine kinase is the enzyme required in the production of the DNA building block
thymidine triphosphate (TTP). It is thus essential for the virus reproduction (40).
Suvaxyn Aujeszky
- 14 -
The glycoprotein E gene and thymidine kinase gene are deleted from Aujeszky's
Disease Virus (41).
Poulvac E. coli
The mutant aroA gene was created by separately polymerase chain reaction (PCR)
amplifying the 5' and 3' portions of the wild type gene (omitting a 100 base pair (bp)
region in the centre of the gene) and ligating these two PCR products together into a
shuttle plasmid. The resultant aroA gene was thereby inactivated by the deletion of the
internal 100 bp region, and also by the incorporation of two stop codons via the
internal PCR primers (48).
Differences between the Biological Characteristics of the GMO and
those of the Recipient Organism or Parental Organism
Vaccines # 1 - 6
The host ranges of the GMOs are expected to be the same as the parental organisms.
The genetic modifications would not enhance the virulence or the ability to survive in
target or non-target species.
PreveNile
The host range of the recombinant vaccine organism appears to be similar to the
backbone vaccine strain, which is also not transmissible by mosquitoes (6).
AviPro® Megan® Vac 1
No differences were observed in the lipopolysaccharide profile of the vaccine
organism compared to the parental S. typhimurium strain, and both bacteria express
the O-antigen. The modified vaccine organism retains the naturally occurring ~91 kb
plasmid present in the parental bacterium. Both bacteria can ferment glucose and
mannose; however, in contrast to wild type S. typhimurium, the vaccine organism is
unable to utilize maltose, mannitol, sorbitol, sucrose, melibiose, rhamnose or xylose
as the sole carbon source, due to the loss-of-function mutations in cya and crp. The
growth rate of the mutant strain on Luria-Bertani broth is slightly reduced compared
to the parental organism. Whereas the parental S. typhimurium strain is virulent to
young chicks (LD50 value of 3x103 CFU), the ∆cya ∆crp S. typhimurium strain has
- 15 -
lost the ability to cause disease, and has an LD50 value estimated to be greater than
4x109 CFU in one-day-old chicks (7, 8).
Poulvac ST
The parent strain infects many species and the mutant vaccine strain can theoretically
do the same. However, the latter cannot persist in vertebrates due to its requirement
for para-aminobenzoic acid (PABA) (35).
Vaxxitek HVT+IBD
The recombinant HVT + IBD virus differs genetically from the parental HVT by the
integration of the expression cassette described in section 4.2. The foreign DNA is
inserted into what is thought to be a non-coding region of the HVT genome, and
hence presumably does not disrupt any endogenous genes. The genetically modified
organism does not contain any selectable markers such as antibiotic resistance genes.
DNA sequencing of the recombinant HVT + IBD virus by the manufacturer
determined that the recombination event between the donor plasmid and wild type
parental HVT was perfectly homologous (10).
Innovax-ILT
The host range, tissue tropism, and shed / spread capabilities of the recombinant
organism are expected to be the same as the parental HVT vaccine strain (37).
Innovax-ND-SB
The host range, tissue tropism, and shed/spread capabilities of the recombinant
organism are expected to be similar to the parental HVT vaccine strain (38)
Nobi-Porvac Aujeszky
The evidence from testing indicated in particular that the genetic modification of the
virus, was not expected to result in any post-release shift in biological interactions or
host range or in any known or predictable effects on non-target organisms in the
environment or other potentially significant interaction with the environment or in any
increase in pathogenicity as compared to the parental virus strain (39).
Suvaxyn Aujeszky
The absence of the thymidine kinase gene in the Aujeszky’s Disease virus increases
- 16 -
the GMO’s safety by reducing its ability to grow. The absence of the glycoprotein E
gene in the virus reduces significantly the multiplication of the vaccine virus in the
central nervous system of pigs (41).
Poulvac E. coli
Due to the mutation of the aroA gene, the modified E. coli in the vaccine shows an
impaired ability to persist in chickens and is consequently non-pathogenic. The
aroA-mutant vaccine strain retains the expression of the surface appendages that have
been shown to be important in the pathogenesis of avian colibacillosis, such as type 1
fimbriae and flagellae, which might aid in the generation of a robust and specific
immune response (48).
Detection and Identification of the GMO
As the DNA sequences involved in the genetic modifications are readily accessible in
the literature, the GMOs can be detected and identified with high sensitivity by
Polymerase Chain Reaction (PCR).
Intended Use of the GMO
The GMOs are used as main active components of the veterinary vaccines for
vaccination against diseases listed in Table 1.
Likely Potential Receiving Environment
Vaccines Using Canarypox Virus ALVAC as the Vector - (Vaccines # 1 - 6)
The parental poxvirus can survive for extended period of time under normal
environmental conditions, in particular when it is associated with dried scab material
or protected from direct sunlight. The recombinant virus is expected to have same
survival characteristics as the parental poxvirus (30).
Vaccine Using Streptococcus equi as the Vector - (Equilis StrepE)
When exposed to natural environment (e.g. under direct exposure to sunlight), the
parental strain can survive for a day or two. The survival of the bacteria may last for a
- 17 -
little bit longer if protected from sunlight or kept at low temperature, but it is probably
still fairly short. The gene deletion is expected to weaken the capability of the
recombinant bacteria to persist in the environment (42).
Vaccine Using Human Yellow Fever Virus as the Vector - (PreveNile)
The recombinant virus can stay alive for prolonged periods at -70oC and 4
oC, but the
viability is greatly reduced at room temperature or above (6).
Vaccines Using Turkey Herpesvirus as the Vector - (Vaxxitek HVT+IBD,
Innovax-ILT and Innovax-ND-SB)
Herpesviruses are destructible by UV light from the sun. But HVT in dried feathers
and poultry dust can remain infectious for up to a year. It is expected that the
recombinant virus should have similar survival characteristics with the parental virus
(10).
AviPro® Megan® Vac 1
The GM bacterium reportedly grows slower than the wild type parental organism, and
has lost the ability to metabolize alternative carbohydrate sources. These defects and
others induced by the gene deletions should hinder the persistence of the vaccine
organism in the environment. It was described by the manufacturer as being sensitive
to the antibiotics amikacin, ampicillin, carbenicillin, cefoxitin, cephalotin,
chloramphenicol, gentamicin, tetracycline, tobramycin, trimethoprim sulfate,
kanamycin, neomycin and streptomycin. The ∆cya ∆crp S. typhimurium should retain
sensitivity to these same antibiotics (7).
Poulvac ST
Since the gene deletion renders it auxotrophic, the vaccine strain cannot survive in
vertebrates or in the environment (35).
Vaccines Using Aujeszky's Disease Virus as the Vector
The parental virus can survive for extended periods under winter conditions below
4oC. But it can be rapidly inactivated at 37
oC in sunlight and in dry conditions. It can
remain infectious at pH5-9, though extreme acidity and alkalinity have deleterious
effects on the virus. Flavoured environments, such as contaminated straw and feeding
- 18 -
troughs, can sustain the virus for 10-30 days at 24oC and for up to 46 days at -20
oC.
The persisting properties of the recombinant virus should be similar to that of the
parental strain (43).
Poulvac E. coli
In back passage studies, the aroA deleted vaccine was unable to survive three back
passages due to the inability to generate p-aminobenzoate (PABA) for continued
survival and generate a self-sustaining population in the environment, thus eliminating
the risk to other vertebrates that may have come in contact with the vaccine (50).
Identification of any Novel Genotypic and Phenotypic Characteristics
Associated with the GMO that may have an Adverse Effect on
Biological Diversity in the Likely Potential Receiving Environment
The potential adverse effects associated with the novel genotypic characteristics of
live recombinant veterinary vaccines may include:
1. Establishing an undesirable self-sustaining population
2. Altered pathogenicity or host range
3. Horizontal gene transfer and recombination with other virus / bacteria
4. Reversion to virulence
5. Possibility to spread to the environment
6. Effects on local host species
Evaluation of the Likelihood of the Adverse Effect Being Realized
Vaccines Using Canarypox Virus ALVAC as the Vector - (Vaccines # 1 - 6)
Establishing an undesirable self-sustaining population
The self-sustaining of the recombinant virus, which means the indefinite survival of
- 19 -
the population by replicating and spreading from host to host, was determined to be
highly improbable (24). These ALVAC-based vaccines were designed to be used in
mammals. As no virus shedding or spreading was detected after vaccination,
non-target animals will not be infected. As a result, the virus is unlikely to replicate
and establish an undesirable population in the non-target animals of the local
environment.
Altered pathogenicity or host range
The safety of several recombinant ALVAC vaccines (Canine Distemper, Feline Rabies,
Feline Leukaemia, Equine Influenza and Equine WNV) has been tested in canary
birds in comparison with the original ALVAC vaccine. Similar tests were conducted
on chicken and mice. Like the parental vaccine, the recombinant viruses cause mild
local lesions at the inoculation sites of the infected canary birds, which were
recovered soon afterwards. No lesion was observed on inoculated chicken and mice
(3).
The safety of various ALVAC®
vaccines was also assessed under laboratory and field
conditions in a variety of species including mice, horses, humans, dogs and chickens.
Safety of ALVAC®
vaccines has been confirmed in animals of a variety of ages with
varying immune statuses and using various routes and doses of administration (44, 45).
It was reported that all the genetically modified viruses tested to date were as safe as
the parental strain and no change of host specificity was observed.
Horizontal gene transfer and recombination with other virus
Molecular interaction between poxvirus within co-infected cells could result in
recombination. Since mammals are not infected by avipoxvirus, in vivo recombination
between the recombinant virus and its wild type relatives can hardly happen.
Although cats can be infected by cowpox virus which could recombine with
avipoxvirus, the chance for the recombinant virus and cowpox virus to exist in the
same cell is extremely low. The wide genetic distance between avipoxvirus and
cowpox virus further minimizes the risk of reverting to virulence by recombination
(46). Thus, it is unlikely for the horizontal gene transfer and recombination to take
place.
- 20 -
Reversion to virulence
Virulence reversion could occur when the non-pathogenic virus recombines with its
virulent relatives or spontaneously mutated during consecutive passages in different
cells. In this case, recombination could hardly take place because the virulent strains
could only be found in avian species but not the inoculation targets (i.e. mammals).
And the recombinant virus is considered to be genetically and phenotypically stable as
no alternation was detected in an experiment with 20 cell culture passages (30). Thus
the virulence reversion is unlikely to happen.
Possibility to spread to the environment
No virus shedding was detected from samples of saliva, urine and faeces collected
from administered kitten (30). Thus, the likelihood of spreading to non-target animals
is very low.
Effects on local host species
Canaries are not native species in Hong Kong. They are imported as pets and kept in
cages. Because the recombinant viruses have the same mild pathogenicity and host
range as the non-recombinant ALVAC virus, they should not impose any threats to the
wild bird species in Hong Kong.
Vaccines Using Streptococcus equi as the Vector - (Equilis StrepE)
Establishing an undesirable self-sustaining population
The attenuation of the recombinant bacteria is achieved by deleting the aroA gene
which is essential for cell replication. It is thus expected that the recombinant bacteria
could not form an undesirable self-sustaining population (47).
Altered pathogenicity or host range
The recombinant vaccine is regarded as non-pathogenic. Based on the nature of the
genetic modification, the recombinant bacteria is unlikely to have its host range
altered (47).
Horizontal gene transfer and recombination with other bacteria
- 21 -
It is possible that the GM bacteria could recombine with wild type S. equi or other
homologous species, but it would only lead to the wild type species that is already
present in the field (34).
Reversion to virulence
Experiments with five and six passages in horses were performed to test the virulence
reversion. In the study using five passages, the deleted aroA gene was not re-acquired
as confirmed by PCR result. In the horses through which the sixth passage was made,
the recombinant vaccine was completely eradicated two weeks after inoculation (34).
Thus the virus should be regarded as genetically and phenotypically stable.
Recombination could take place only if the horse is already contracted with the wild
type bacteria species, leading only to the normal wild type S. equi already present in
the field.
Possibility to spread to the environment
It was demonstrated that the vaccine strain does not spread from vaccinated horses to
in-contact horses (34). And because the recombinant bacteria do not infect animals
other than equine species, the risk for the vaccine strain to spread to the environment
is negligible.
Effects on local host species
Hong Kong has no native equine species. Horses that are to be imported are required
to go through a series of inspection and quarantine measures to ensure their
healthiness. Thus, no local host species would be affected.
Vaccines Using Human Yellow Fever Virus as the Vector - (PreveNile)
Establishing an undesirable self-sustaining population
According to studies, the replication of the recombinant virus is very limited in the
administered horses. The virus will not be transmitted to animals other than the
intended targets, as no virus shedding or spreading was detected after vaccination. The
recombinant virus is also not transmissible by mosquitoes (6). Thus, the virus is
unlikely to establish an undesirable self-sustaining population.
- 22 -
Altered pathogenicity or host range
There have been in vivo and in vitro studies showing that the virulence properties and
host range specificity of the recombinant virus are similar to those of the backbone
Yellow Fever Virus strain (6).
Horizontal gene transfer and recombination with other virus
Intertypic recombination of Dengue Virus, which belongs to the same Flaviviridae
family as the parental Yellow Fever Virus, was observed infrequently during extended
periods of high viremia in humans. Interspecific recombination has not been reported
in the literature. Since the virus has very limited replication in the horses and the
viremia following West Nile Vrius infection is typically of low titre and short duration,
recombination between West Nile Virus and the recombinant vaccine is unlikely to
take place (6).
Reversion to virulence
Back passage attempts have demonstrated the genetic and phenotypic stability of the
recombinant virus (6). The recombinant virus is unlikely to revert to virulence.
Possibility to spread to the environment
The recombinant virus has no shedding in horses and thus is not likely to spread to the
environment (6).
Effects on local host species
Human is susceptible to the wild type Yellow Fever Virus. This recombinant virus is
regarded to be as safe to human health as the backbone Yellow Fever vaccine strain,
which has been widely used in the world since 1937 (6).
Vaccines Using Turkey Herpesvirus as the Vector - (Vaxxitek HVT+IBD,
Innovax-ILT and Innovax-ND-SB)
Establishing an undesirable self-sustaining population
The parental virus can replicate in cells of avian origin (particularly chicken, turkey,
duck and quail). The shedding of the virus can be detected in feather dander. However,
it is not reported that the Turkey Herpesvirus can spread between chicken, given that
- 23 -
the virus has been used as vaccine against Infectious Bursal Disease since 1970s. Data
submitted by the manufacturer showed that the recombinant virus will not spread
from vaccinated chickens to in-contact chickens. However, the recombinant virus, like
the parental virus, can spread from vaccinated chickens to in-contact turkeys (10).
Altered pathogenicity or host range
Since only a gene with known function is added and the insertion site is at a
non-coding region of the HVT genome, it is expected that the genetic modification
would not alter the recombinant vaccine’s pathogenicity or host range (10).
Horizontal gene transfer and recombination with other virus
In experiments performed by the manufacturer, the recombinant virus was inoculated
to chickens together with either a serotype 1 or serotype 2 MD virus. The infected
chickens were not sick or killed by the infection. In addition, the parental HVT has
been widely used as a component of bivalent vaccines with other MD viruses with
high safety over the past 30 years. These two events indicate that either there is no
recombination or the recombination does not result in producing pathogenic virus (10).
As the parental virus could latently infect avian cells and integrate into host
chromosome for prolonged periods, recombination with other viruses can
theoretically take place.
Reversion to virulence
A back passage study with nine passages in chickens was performed by the
manufacturer and showed no increase in morbidity or mortality. Thus, the
recombinant virus is considered to be phenotypically and genetically stable. Reversion
to virulence due to recombination has not been observed on the parental HVT (10).
Possibility to spread to the environment
If the poultry dust from farms rearing vaccinated chickens is not properly disposed,
the recombinant virus could spread to quails or other birds in Hong Kong.
Effects on local host species
The virus is shed in feather dander and was demonstrated capable of spreading from
- 24 -
vaccinated chickens to in-contact turkeys. Since quails are also susceptible to HVT,
the recombinant virus may spread to these birds. There are three quail species:
Coturnix japonica (Japanese Quail), Turnix suscitator (Barred Button-quail) and
Turnix tanki (Yellow-legged Button-quail) found in Hong Kong, and theoretically
they are susceptible to the recombinant virus. However, as the vaccine is
non-pathogenic, the risk to the wild quail species in Hong Kong is considerable very
low.
Vaccines Using Salmonella typhimurium as the Vector - (AviPro® Megan® Vac 1
and Poulvac ST)
Establishing an undesirable self-sustaining population
Deletion of the aroA, cya and crp genes impairs the essential cellular functions of the
recombinant bacteria. As a result, the bacteria grow more slowly and are incapable to
invade and infect hosts. Therefore, it is unlikely for the recombinant bacteria to
establish an undesirable self-sustaining population (7).
Altered pathogenicity or host range
The pathogenicity of the recombinant bacteria is diminished. Its host range should not
be different from that of the parental bacteria.
Horizontal gene transfer and recombination with other bacteria
The parental bacteria are known to acquire genes through horizontal gene transfer.
The recombinant bacteria should have similar tendency in carrying out recombination.
Reversion to virulence
In the experiments with successive passages in chicks conducted by the manufacturer,
no gross genetic changes were detected in the DNA surrounding the deletions. It
thus confirmed the genetic and phenotypic stability of the two recombinant bacteria.
Reversion to virulence of the double mutant (AviPro® Megan® Vac 1, ∆cya and ∆crp)
by recombination is considered not feasible. The locations of the two genes on the
chromosome are separated at great distance. One single recombination event cannot
cover the entire section and double recombination in the same cell is extremely rare
(7).
- 25 -
On the other hand, it is rare but still possible for the aroA mutant (Poulvac ST) to
acquire functional aroA allele from wild type species by recombination and regain
virulence. But it only leads to the wild type species that is already present in the field.
Possibility to spread to the environment
The recombinant bacteria were continuously shed by administered birds for up to 13
weeks post-inoculation. They also appear capable of spreading to in-contact birds (7).
Effects on local host species
A variety of species found in Hong Kong, such as horses, cattle, pigs, dogs and cats,
are vulnerable to S. typhimurium infection. The wild relatives, such as Prionailurus
bengalensis (Leopard Cat) might as well be susceptible to the infection. Humans are
also potential host to this bacterial pathogen and thus can become carriers to spread
the bacteria to local host species. However, since the vaccines are non-pathogenic, the
risk to the wild mammal species in Hong Kong is very low.
Vaccines Using Aujeszky's Disease Virus as the Vector
Establishing an undesirable self-sustaining population
The deletion of the essential genes, particularly the thymidine kinase that is required
for DNA synthesis, would diminish the recombinant virus’ ability to grow and infect
the hosts. It is thus expected that the recombinant virus cannot establish a
self-sustaining population (41, 43).
Altered pathogenicity or host range
The pathogenicity of the recombinant viruses is diminished. Their host range should
not be different from that of the parental virus strains.
Horizontal gene transfer and recombination with other virus
It was justified that the intertypic recombination of the vaccine virus with other
Aujeszky's Disease Virus is rare and the risk is considered to be acceptable (43).
Reverting to virulence
- 26 -
Experiments showed that the recombinant viruses did not revert to virulence and
could not be recovered after 3 or 4 passages (43).
Possibility to spread to the environment
It was reported that the recombinant viruses did not spread from vaccinated pigs to
in-contact susceptible pigs (43).
Effects on local host species
Though the parental virus can infect nearly all domesticated and wild mammals
including cattle, sheep, goats, cats and dogs (11), the recombinant viruses are
attenuated and have very limited replication in the hosts and thus it is unlikely that
they would have any significant effects on local non-target species.
Vaccines Using E. coli as the Vector - (Poulvac E. coli)
Establishing an undesirable self-sustaining population
The aroA defect means that the vaccine E. coli strain cannot survive without
supplemental aromatic amino acids or PABA, which are not readily available in the
environment (48).
Altered pathogenicity or host range
Based on the nature of the genetic modification, the recombinant bacteria is unlikely
to have its host range altered.
Horizontal gene transfer and recombination with other bacteria
E. coli are known to acquire foreign DNA through horizontal gene transfer, via
conjugation (typically only plasmid DNA transferred), by transduction (involving
bacteriophage), or by free DNA uptake (transformation). However, such a horizontal
gene transfer event would not increase the bacteria's pathogenicity beyond that of the
wild type parental E. coli, which may already be present at the farm, or not unlike
other pathogenic E. coli in the poultry flock being vaccinated. Further, the chances of
horizontal gene transfer taking place after vaccination is significantly restricted, due
to the inability of the vaccine organism to persist in an environment devoid of its
requisite aromatic nutrients (48).
- 27 -
Reversion to virulence
The manufacturer estimates that the reversion rate of the vaccine strain back to the
parental strain is less than 10-11
for the aroA mutation. Deletion of a large part of a
gene provides confidence that random spontaneous mutations will be unable to repair
the loss of function, especially compared to a system of inactivation dependent on the
modification of only a few nucleotides. The genetic stability of the master seed under
normal culture conditions has been demonstrated up to n+5 passages, which is the
upper limit of fermentation specified for the production of the vaccine (48).
Possibility to spread to the environment
In a shed/spread study conducted by the manufacturer, swabs were taken from the
feed bins, water bowls, floor, and top-front part of the cages following the vaccination
of fifty chicks by coarse spray to monitor environment contamination by the vaccine
organism. The vaccine organism was recovered from the environment at 3 and 7 days
post-vaccination, but not after 10, 14, or 24 days, confirming the limited persistence
of the aroA mutant E. coli.
Effects on local host species
A lot of species in Hong Kong are susceptible to E. coli infection. However, since the
vaccine is non-pathogenic, the risk to the wild species in Hong Kong is very low.
Evaluation of the Consequences should the Adverse Effect be
Realized
Establishing an undesirable self-sustaining population
As the vaccines are not pathogenic, establishment of an undesirable
self-sustaining population should not result in significant adverse effect on Hong
Kong’s biodiversity.
Altered pathogenicity or host range
Since the genes modified are not relevant to the pathogenicity, it is speculated
that the genetic modification would not enhance the pathogenicity. Even if
pathogenicity is changed, it shall not result in significant adverse effect on Hong
Kong’s biodiversity. On the other hand, if the host range is changed, there might
- 28 -
be adverse effects on Hong Kong’s biodiversity as the pathogenicity on different
hosts may be different.
Horizontal gene transfer and recombination with other viruses / bacteria
If horizontal gene transfer and recombination with other viruses / bacteria occur,
the chance to produce viral or bacterial strains with severe pathogenicity shall
not be different from those happening among non-GM strains.
Reversion to virulence
As the diseases are already present in nature, the reversion to virulence shall not
result in significant adverse effect on Hong Kong’s biodiversity.
Possibility to spread to the environment
As the target animals are all domesticated, spreading to the environment would
be limited. And as the diseases are already present in nature, the spreading of the
non-pathogenic vaccine strains to the environment shall not result in any
significant adverse effect on Hong Kong’s biodiversity.
Effects on local host species
As the vaccines are not pathogenic, adverse effect on local host species is not
anticipated.
Estimation of the Overall Risk Posed by the GMO
Vaccines Using Canarypox Virus ALVAC as the Vector - Vaccines # 1 - 6
Based on the above risk assessment, it is concluded that the potential risk of
ALVAC-based recombination vaccines to biodiversity is low and acceptable.
Vaccines Using Streptococcus equi as the Vector - (Equilis StrepE)
Based on the above risk assessment, it is concluded that the potential risk of S.
equi-based recombination vaccine to biodiversity is low and acceptable. The current
inspection and quarantine measures imposed on imported horses should be adequate
for preventing the virulent strain of the bacteria from entering into Hong Kong. It thus
- 29 -
reduces the risk of reversion to virulence by recombination.
Vaccines Using Human Yellow Fever Virus as the Vector - (PreveNile)
Based on the above risk assessment, it is concluded that the potential risk of Human
Yellow Fever Virus-based recombination vaccine to biodiversity is low and
manageable.
Vaccines Using Turkey Herpesvirus as the Vector - (Vaxxitek HVT+IBD,
Innovax-ILT and Innovax-ND-SB)
The viruses could be shed by vaccinated chicks and persist in the environment in dust
for prolonged periods. However, the pathogenicity to wild bird species in Hong Kong
is considered acceptable. The overall risks are thus considered low and manageable.
Vaccines Using Salmonella typhimurium as the Vector - (AviPro® Megan® Vac 1
and Poulvac ST)
The only biosafety risk that theoretically may be expected is the recombination with
wild type S. typhimurium, which would result only in the presence of the normal wild
type bacteria that is already present. Thus the vaccines’ risk to the local biological
diversity is considered low and manageable.
Vaccines Using Aujeszky's Disease Virus as the Vector - (Suvaxyn Aujeszky and
Nobi-Porvac Aujeszky)
Based on the properties of the vaccines, it is considered that the potential risk of
Aujeszky's Disease Virus-based recombination vaccines to biodiversity is low and
acceptable.
Vaccine Using E. coli as the Vector - (Poulvac E. coli)
In view of the fact that the vaccine strain was rendered non-pathogenic, it is
considered the potential risk of the recombination vaccine to biodiversity is low.
June 2011
Agriculture, Fisheries and Conservation Department
- 30 -
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